1. Field of the Invention
The invention relates to pyrazolopyrrolidine compounds, which inhibit gamma secretase and β-amyloid peptide release and/or its synthesis. Therefore, the compounds of the present invention are useful in the prevention and/or treatment of cognitive disorders in patients susceptible to cognitive disorders, and more specifically in preventing, treating, and/or halting the progress of neurodegenerative disorders such as Alzheimer's disease, dementia, mild cognitive impairment, dementia, Down's syndrome, and other similar diseases. The compounds of the invention are also useful for initiating or increasing angiogenesis.
2. State of the Art
Alzheimer's Disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and ultimately death.
The brains of individuals with AD exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles. Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles, are generally found in several areas of the human brain important for memory and cognitive function in patients with AD. Smaller numbers of these lesions in a more restrictive anatomical distribution are also found in the brains of most aged humans who do not have clinical AD. Amyloid plaques and amyloid angiopathy also characterize the brains of individuals with Trisomy 21 (Down's Syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch Type (HCHWA-D). At present, a definitive diagnosis of AD usually requires observing the aforementioned lesions in the brain tissue of patients who have died with the disease or, rarely, in small biopsied samples of brain tissue taken during an invasive neurosurgical procedure.
The principal chemical constituent of the amyloid plaques and vascular amyloid deposits (amyloid angiopathy) characteristic of AD and the other disorders mentioned above is an approximately 4.2 kilodalton (kD) protein of about 38-43 amino acids designated the β-amyloid peptide (βAP) or sometimes Aβ, AβP or β/A4. Glenner, G. G. et al (Biochem. Biophys. Res. Commun., 120:885-890 (1984) first purified the β-Amyloid peptide and provided a partial amino acid sequence. The isolation procedure and the sequence data for the first 28 amino acids are described in U.S. Pat. No. 4,666,829.
Molecular biological and protein chemical analyses have shown that the β-amyloid peptide is a small fragment of a much larger precursor protein termed the amyloid precursor protein (APP), that is normally produced by cells in many tissues of various animals, including humans. Knowledge of the structure of the gene-encoding APP has demonstrated that β-amyloid peptide arises as a peptide fragment that is cleaved from APP by protease enzyme(s). Sequential processing of the precursor protein by the enzymes referred to generically as beta- and gamma-secretases, give rise to the β-amyloid peptide fragment. Both enzymes have now been molecularly cloned, and characterized to differing levels.
Several lines of evidence indicate that progressive cerebral deposition of β-amyloid peptide plays a seminal role in the pathogenesis of AD and can precede cognitive symptoms by years or decades. See, for example, Selkoe, D J. Neuron, 6:487-498 (1991). The most important line of evidence is the discovery that missense DNA mutations at amino acid 717 of the 770-amino acid isoform of APP can be found in affected members but not unaffected members of several families with a genetically determined (familial) form of AD (Goate, A. et al., Nature, 349:704-706 (1991); Chartier-Harlin, M. C. et al., Nature, 353:844-846 (1991); and Murrell, J. et al., Science, 254:97-99 (1991.) Another such mutation, known as the Swedish variant, is comprised of a double mutation changing lysine595-methionine596 to asparagine595-leucine596 (with reference to the 695 isoform was found in a Swedish family) was reported in 1992 (Mullan, M. et al., Nature Genet., 1:345-347 (1992). Genetic linkage analyses have demonstrated that these mutations, as well as certain other mutations in the APP gene, are the specific molecular cause of AD in the affected members of such families. In addition, a mutation at amino acid 693 of the 770-amino acid isoform of APP has been identified as the cause of the β-amyloid peptide deposition disease, HCHWA-D, and a change from alanine to glycine at amino acid 692 appears to cause a phenotype that resembles AD is some patients but HCHWA-D in others. The discovery of these and other mutations in APP in genetically based cases of AD prove that alteration of APP metabolism, and subsequent deposition of its β-amyloid peptide fragment, can cause AD.
Despite the progress which has been made in understanding the underlying mechanisms of AD and other β-amyloid peptide related diseases, there remains a need to develop methods and compositions for treatment of the disease(s). Ideally, the treatment methods would advantageously be based on drugs, which are capable of inhibiting β-amyloid peptide release and/or its synthesis in vivo.
One approach toward inhibiting amyloid peptide synthesis in vivo is by inhibiting gamma secretase, the enzyme responsible for the carboxy-terminal cleavage resulting in production of β-amyloid peptide fragments of 40 or 42 residues in length. The immediate substrates for gamma secretase are β-cleaved, as well as α-cleaved carboxy-terminal fragments (CTF) of APP. The gamma-secretase cleavage site on β- and α-CTF fragments occurs in the predicted transmembrane domain of APP. Inhibitors of gamma-secretase have been demonstrated to effect amyloid pathology in transgenic mouse models (Dovey, H. F, et al. “Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain.” J Neurochem 76 (1): 173-81 (2001))
Gamma secretase is recognized to be a multi-subunit complex comprised of the presenilins (PS1 or PS2), Nicastrin, Aph-1, and Pen 2 (De Strooper, B. “Aph-1, Pen-2, and Nicastrin with Presenilin generate an active gamma-Secretase complex.” Neuron 38(1): 9-12 (2003); Edbauer, D., et al. “Reconstitution of gamma-secretase activity.” Nat Cell Biol 5 (5): 486-8; (2003); Kimberly, W. T et al. “Gamma-secretase is a membrane protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2.” Proc Natl Acad Sci USA 100 (11) 6382-7 (2003)). Much evidence indicates that PS comprises the catalytic moiety of the complex, while the other identified subunits are necessary for proper maturation and sub-cellular localization of the active enzyme complex (reviewed in De Strooper, B. “Aph-1, Pen-2, and Nicastrin with Presenilin generate an active gamma-Secretase complex.” Neuron 38 (1): 9-12.) (2003). Consistent with this hypothesis: PS knock-out mice exhibit significant reductions in β-amyloid production (De Strooper. B et al., “Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein.” Nature 391(6665): 387-90 (1998); Haass, C. et al. “Alzheimer's disease. A technical KO of amyloid-beta peptide.” Nature 391 (6665) 339-40 (1998); Herreman, A., L et al. “Total inactivation of gamma-secretase activity in presenilin-deficient embryonic stem cells.” Nat Cell Biol 2(7): 461-2 (2000)); point mutations of putative active site aspartate residues in PS trans-membrane domains inhibit β-amyloid production in cells in a dominant negative fashion (Wolfe, M. S. et al. “Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity.” Nature 398 (6727): 513-7 (1999); Kimberly, W. T. et al. “The transmembrane aspartates in presenilin 1 and 2 are obligatory for gamma-secretase activity and amyloid beta-protein generation.” J Biol Chem 275(5): 3173-8 (2000)); active site directed substrate-based transition state isosteres designed to inhibit gamma secretase directly conjugate to PS (Esler, W. P., et al. “Transition-state analogue inhibitors of gamma-secretase bind directly to presenilin-1.” Nat Cell Biol 2(7): 428-34 (2000); Li, Y. M., et al “Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1.” Nature 405(6787): 689-94 (2000).); finally, allosteric gamma secretase inhibitors have likewise been demonstrated to bind directly to PS (Seiffert, D., et al. “Presenilin-1 and -2 are molecular targets for gamma-secretase inhibitors” J Biol Chem 275 (44) 34086-91 (2000)).
Current evidence indicates that in addition to APP processing leading to β-amyloid synthesis, gamma-secretase also mediates the intra-membrane cleavage of other type I transmembrane proteins (reviewed in Fortini, M. E. “Gamma-secretase-mediated proteolysis in cell-surface-receptor signaling.” Nat Rev Mol Cell Biol 3 (9): 673-84 (2002), see also Struhl, G. et al. “Requirements for presenilin-dependent cleavage of notch and other transmembrane proteins.” Mol Cell 6(3): 625-36. (2000).) Noteworthy among the known substrates of gamma-secretase is mammalian Notch 1. The Notch 1 protein is important for cell fate determination during development, and tissue homeostasis in the adult. Upon ligand engagement via the Notch ecto-domain, Notch undergoes sequential extra-cellular and intra-membrane processing analogous to APP. The intra-membrane processing of Notch mediated by gamma secretase leads to release of the Notch intracellular domain (NICD). The NICD fragment mediates Notch signaling via translocation to the nucleus, where it regulates expression of genes mediating cellular differentiation in many tissues during development, as well as in the adult.
Disruption of Notch signaling via genetic knock-out (KO) results in embryonic lethal phenotype in mice (Swiatek, P. J et al. “Notch1 is essential for postimplantation development in mice.” Genes Dev 8 (6): 707-19 (1994) and Conlon, R. A et al. “Notch1 is required for the coordinate segmentation of somites” Development 121 (5): 1533-45 (1995)). The Notch KO phenotype is very similar to the phenotype observed PS1 KO mice, and precisely reproduced by PS1/PS2 double KO mice (De Strooper et al. “Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein.” Nature 391(6665): 387-90 (1998); Donoviel, D. B., et al. “Mice lacking both presenilin genes exhibit early embryonic patterning defects.” Genes Dev 13(21): 2801-10 (1999); and Herreman, A., et al “Total inactivation of gamma-secretase activity in presenilin-deficient embryonic stem cells” Nat Cell Biol 2 (7): 461-2 (2000)).
Gamma-secretase inhibitors have also been shown to increase angiogenesis. See US 2006/0264380. As such, the gamma secretase inhibitors of the invention are useful in promoting angiogenesis.
Other studies have found that gamma-secretase inhibitors can prevent Notch activation and reduce proliferation in some human cancers. Konishi J, et al., Cancer Res. 2007 Sep. 1; 67(17):8051-7; Miele L, et al., Curr Cancer Drug Targets. 2006 June; 6(4):313-23, “NOTCH signaling as a novel cancer therapeutic target”.